Abstract Arenaviruses such as Lassa fever virus (LASV) can cause acute viral hemorrhagic fever disease in humans to which there is no vaccine or effective treatment. The mechanism of arenavirus replication in the infected cells is poorly understood, which has impeded the development of effective antivirals and vaccines. Arenaviral mRNA contains several nontemplated nucleotides that are believed to derive from the host cell messenger RNAs through the cap-snatching mechanism. To date, little is known about the specific protein(s) and the molecular mechanism that are involved in the cap-snatching process for arenaviruses. We have recently obtained a 3-D crystal structure of the first arenavirus protein, the LASV nucleoprotein NP, and identified several novel structural motifs that suggest NP as the first arenaviral protein involved in the cap-snatching process. We hypothesize that NP is an essential component of viral replication complex that functions to steal the 5[unreadable] cap structures of cellular mRNA in order to initiate viral transcription. The main goal of this study, therefore, is to combine our strengths in molecular virology, structural modeling, and RNA/protein biochemistry and the availability of several novel viral reverse genetics systems in our laboratory to clearly define the molecular mechanism of cap snatching in arenaviruses. Specifically, we will characterize the cap-snatching function of the LASV NP protein in vitro by biochemical analyses (Aim 1) and in vivo by several novel and functional cell-based assays and infectious virus systems (Aim 2). Lastly, we wish to characterize the molecular interactions between the viral NP and L proteins within the replication complex in mediating and/or regulating the cap-snatching process (Aim 3). Taken together, these studies will reveal for the first time a novel molecular mechanism of the cap-snatching step for arenavirus and its biological significance in transcribing the viral genome in vitro and in vivo. These studies will shed important lights on arenaviral RNA transcription that may offer opportunities for development of effective therapeutics and/or vaccines against lethal arenaviral hemorrhagic fever infection in humans.